The theory of survival of the fittest has long been the backbone of species’ evolutionary adaptations. Survival struggles, as the main behavioral response of species adapting to their environment, have played a critical role throughout the long course of biological evolution. In the face of natural threats, both humans and animals have evolved a series of relatively conserved inter-species survival struggle behaviors. The pioneering research by Fanselow and colleagues proposed the classic theoretical model “Predatory Imminence Continuum” [1], which describes the relationship between inter-species survival behaviors and the “prey-predator” distance. The model indicates that when prey is far from a predator, the prey mainly adopts defensive strategies like freezing or fleeing. However, when a predator is in close proximity and escape is no longer possible, the prey must resort to direct confrontation and enter into an offensive defense mode. Defensive attack behavior is the last line of defense for prey against predators, and the neural mechanisms of this critical survival behavior have long remained an unresolved scientific issue.
On January 3, 2022, the research team of Peng Cao at the Beijing Institute of Life Sciences / Tsinghua University Joint Biomedical Research Institute published a study titled Mechanically evoked defensive attack is controlled by GABAergic neurons in the anterior hypothalamic nucleus in the prestigious neuroscience journal Nature Neuroscience. The study revealed that when prey is cornered and unable to escape, harmful mechanical stimuli serve as a key trigger for defensive attack behavior, and GABAergic neurons in the anterior hypothalamic nucleus (AHN) are crucial in encoding these harmful mechanical stimuli. This study not only elucidates the neural mechanisms of instinctive defensive attack behavior but also highlights the important role of pain perception induced by harmful mechanical stimuli in the inter-species survival struggle.
1. Harmful Mechanical Stimuli as a Key Trigger for Defensive Attack Behavior
The researchers first conducted a series of behavioral experiments with C57BL/6 mice to identify the key stimuli that trigger defensive attack behaviors. First, they applied the odor of a snake to a simulated snake to provide a sensory cue of the predator or connected a crocodile clip to apply harmful mechanical stimuli. They found that the simulated snake with predator odor did not induce defensive attack behavior, but when a crocodile clip applied continuous harmful mechanical stimuli to the mouse’s tail, the mice attacked the simulated snake with biting behavior. This attack behavior also occurred in the dark, suggesting that vision was not involved in triggering this behavior. Second, when the crocodile clip was connected to harmless objects (such as a plastic bottle cap or wooden block), it still triggered defensive attack behavior when the clip applied mechanical stimuli to the mouse’s tail. These results show that it is not the object connected to the crocodile clip that is important, but the harmful mechanical stimuli it provides. Third, using the Cre enzyme-dependent diphtheria toxin (iDTR-DT) system, the researchers killed Mrgprd+ neurons in the dorsal root ganglion (DRG) via diphtheria toxin (DTX) injection, which significantly weakened defensive attack behaviors. Mrgprd+ neurons in the DRG are a type of sensory neuron sensitive to harmful mechanical stimuli, further demonstrating that mechanical stimuli can trigger defensive attack behavior through these sensory neurons. Fourth, the mechanical stimulus-induced defensive attack behavior showed no gender differences. Fifth, by altering the weight of the simulated snake and the experimental environment’s area, the researchers found that the “escape potential” in the defensive scenario could also be a determining factor for whether mechanical stimuli triggered defensive attack behavior. Overall, these findings indicate that harmful mechanical stimuli are a key trigger for defensive attack behavior when escape is not possible.
2. vGAT+ AHN Neurons are Essential for Triggering Defensive Attack Behavior
The researchers next explored the central neural mechanisms involved in this behavior. They used fluorescence in situ hybridization (FISH) to examine the types of neurons in the AHN and found that vGAT+ neurons predominated. To deactivate these neurons, the researchers combined the Cre-LoxP system with inhibitory optogenetics (GtACR1). In vGAT-IRES-Cre mice, they injected AAV-DIO-GtACR1-2A-EGFP into the AHN and implanted optical fibers to induce specific expression of GtACR1 in vGAT+ neurons. When the simulated snake, attached to a crocodile clip, bit the mouse’s tail to provoke an attack, the continuous blue light suppressed the activity of vGAT+ AHN neurons. Compared to control mice, the experimental mice showed significantly reduced attack frequency and duration, suggesting that vGAT+ AHN neurons are necessary for triggering defensive attack behavior in response to harmful mechanical stimuli.
3. vGAT+ AHN Neurons Encode the Intensity and Location of Mechanical Stimuli
To understand the connection between vGAT+ AHN neurons and harmful mechanical stimuli, the researchers used calcium imaging with optical fibers to record calcium signals in vGAT+ AHN neurons of vGAT-IRES-Cre mice. When the mouse’s tail was bitten by the crocodile clip, the calcium signals from vGAT+ AHN neurons increased significantly. In other scenarios (such as free exploration), these neurons showed only slight increases in calcium signals. Moreover, when the mouse was attacked by a CD1 mouse in a social setting, vGAT+ AHN neurons also showed a significant rise in calcium signals. These results suggest that vGAT+ AHN neurons preferentially respond to harmful mechanical stimuli.
Further, the researchers performed single-unit recordings of vGAT+ AHN neurons using “optical electrodes” and found that these neurons were particularly responsive to harmful mechanical stimuli from the crocodile clip, while their response to the odor of snakes was weak. Additionally, they discovered that vGAT+ AHN neurons encoded different intensities of mechanical stimuli applied to the contralateral side of the body.
4. Upstream Inputs to vGAT+ AHN Neurons
To understand the sources of the input to vGAT+ AHN neurons, the researchers used rabies virus tracing to map the upstream projection network. They found that vGAT+ AHN neurons received input from brain regions involved in processing pain, such as the lateral parabrachial nucleus (LPB) and the paraventricular thalamic nucleus (PVT). They also found that these neurons received input from hypothalamic regions related to aggression (e.g., the ventromedial hypothalamus), regions encoding predator odor (e.g., the medial amygdala), and other regions including the posterior hypothalamic nucleus and the hippocampus. These results helped reconstruct a defensive attack-related neural network centered on vGAT+ AHN neurons.
5. Optogenetic Activation of vGAT+ AHN Neurons Triggers Defensive Attack Behavior
The researchers further tested whether vGAT+ AHN neurons are sufficient to trigger defensive attack behavior using optogenetics. In vGAT-IRES-Cre mice, they injected AAV-DIO-ChR2-mCherry into the AHN and implanted optical fibers. Upon activation of vGAT+ AHN neurons with light, the mice launched defensive attacks against both the simulated snake and a real snake. Importantly, they did not attack social companions. Optogenetic activation of these neurons also suppressed social aggression, proving that vGAT+ AHN neurons are both necessary and sufficient to trigger defensive attack behavior in response to harmful mechanical stimuli.
6. The vGAT+ AHN-vlPAG Pathway is Sufficient to Trigger Defensive Attack Behavior
To investigate the neural circuitry underlying the regulation of defensive attack behavior by vGAT+ AHN neurons, the researchers used the SynaptoTag anterograde tracing method to map the downstream projections of vGAT+ AHN neurons. They found that these projections included regions such as the medial preoptic area (MPA), lateral septum (LS), and the ventromedial hypothalamus (VMH), with specific projections to the ventrolateral periaqueductal gray (vlPAG). Previous research has shown that the vlPAG and LS are involved in aggression-related behaviors. The researchers then compared the roles of the vGAT+ AHN-vlPAG and vGAT+ AHN-LS pathways. They found that activation of the vGAT+ AHN-vlPAG pathway effectively triggered defensive attack behavior, whereas activation of the vGAT+ AHN-LS pathway did not. Using both optogenetic and chemogenetic methods to suppress the vGAT+ AHN-vlPAG pathway, the researchers showed that inhibiting this pathway significantly blocked defensive attack behavior.
These findings demonstrate that the activation of the vGAT+ AHN-vlPAG pathway is both necessary and sufficient to trigger defensive attack behavior in mice.
The study was authored by Zhi-Yong Xie, Hua-Ting Gu, and Mei-Zhu Huang, with other key contributions from lab members (Xinyu Cheng, Cong-Ping Shang, Ting Tao, Da-Peng Li). Collaborative support came from laboratories including those of Zhi-Bin Zhang (Chinese Academy of Sciences), Fan Zhang (Hebei Medical University), Zong-Xiang Tang (Nanjing University of Traditional Chinese Medicine), and Cheng Zhang (Beijing Institute of Life Sciences). The research was funded by the Beijing Institute of Life Sciences and the National Natural Science Foundation of China.
Paper Link: https://www.nature.com/articles/s41593-021-00985-4
References:
- Fanselow, M. S. & Lester, L. S. A functional behavioristic approach to aversively motivated behavior: Predatory imminence as a determinant of the topography of defensive behavior. Evolution and Learning, 185-211 (1988).